EHV-1 glycoprotein D (EHV-1 gD) is required for virus entry and cell-cell fusion, and an EHV-1 gD deletion mutant induces a protective immune response in mice

The viral fusion protein glycoprotein B (gB) is conserved in all herpesviruses and is essential for virus entry. During entry, gB fuses viral and host cell membranes by refolding from a prefusion to a postfusion form. We previously introduced three structure-based mutations (gB-I671A/H681A/F683A) into the domain V arm of the gB ectodomain that resulted in reduced cell-cell fusion. A virus carrying these three mutations (called gB3A) displayed a small plaque phenotype and remarkably delayed entry into cells. To identify mutations that could counteract this phenotype, we serially passaged the gB3A virus and selected for revertant viruses with increased plaque size. Genomic sequencing revealed that the revertant viruses had second-site mutations in gB, including E187A, M742T, and S383F/G645R/V705I/V880G. Using expression constructs encoding these mutations, only gB-V880G was shown to enhance cell-cell fusion. In contrast, all of the revertant viruses showed enhanced entry kinetics, underscoring the fact that cell-cell fusion and virus-cell fusion are different. The results indicate that mutations in three different regions of gB (domain I, the membrane proximal region, and the cytoplasmic tail domain) can counteract the slow entry phenotype of gB3A virus. Mapping these compensatory mutations to prefusion and postfusion structural models suggests sites of intramolecular functional interactions with the gB domain V arm that may contribute to the gB fusion function. Importance The nine human herpesviruses are ubiquitous and cause a range of disease in humans. Glycoprotein B (gB) is an essential viral fusion protein that is conserved in all herpesviruses. During host cell entry, gB mediates virus-cell membrane fusion by undergoing a conformational change. Structural models for the prefusion and postfusion form of gB exist, but the details of how the protein converts from one to the other are unclear. We previously introduced structure-based mutations into gB that inhibited virus entry and fusion. By passaging this entry-deficient virus over time, we selected second-site mutations that partially restore virus entry. The location of these mutations suggest regulatory sites that contribute to fusion and gB refolding during entry. gB is a target of neutralizing antibodies and defining how gB refolds during entry could provide a basis for the development of fusion inhibitors for future research or clinical use.

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Using Antibodies and Mutants To Localize the Presumptive gH/gL Binding Site on Herpes Simplex Virus gD

Journal of Virology ◽

10.1128/jvi.01694-18 ◽

2018 ◽

Vol 92 (24) ◽

Cited By ~ 6

Author(s):

Doina Atanasiu ◽

Wan Ting Saw ◽

Eric Lazear ◽

J. Charles Whitbeck ◽

Tina M. Cairns ◽

...

Keyword(s):

Cell Fusion ◽

Receptor Binding ◽

Protein Interactions ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Epitope Mapping ◽

Virus Entry ◽

Cellular Receptor ◽

Functional Sites ◽

Cell Cell

ABSTRACTHSV virus-cell and cell-cell fusion requires multiple interactions between four essential virion envelope glycoproteins, gD, gB, gH, and gL, and between gD and a cellular receptor, nectin-1 or herpesvirus entry mediator (HVEM). Current models suggest that binding of gD to receptors induces a conformational change that leads to activation of gH/gL and consequent triggering of the prefusion form of gB to promote membrane fusion. Since protein-protein interactions guide each step of fusion, identifying the sites of interaction may lead to the identification of potential therapeutic targets that block this process. We have previously identified two “faces” on gD: one for receptor binding and the other for its presumed interaction with gH/gL. We previously separated the gD monoclonal antibodies (MAbs) into five competition communities. MAbs from two communities (MC2 and MC5) neutralize virus infection and block cell-cell fusion but do not block receptor binding, suggesting that they block binding of gD to gH/gL. Using a combination of classical epitope mapping of gD mutants with fusion and entry assays, we identified two residues (R67 and P54) on the presumed gH/gL interaction face of gD that allowed for fusion and viral entry but were no longer sensitive to inhibition by MC2 or MC5, yet both were blocked by other MAbs. As neutralizing antibodies interfere with essential steps in the fusion pathway, our studies strongly suggest that these key residues block the interaction of gD with gH/gL.IMPORTANCEVirus entry and cell-cell fusion mediated by HSV require gD, gH/gL, gB, and a gD receptor. Neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with an essential step in the fusion pathway. Thus, the epitopes of these MAbs identify critical, functional sites on their target proteins. Unlike many anti-gD MAbs, which block binding of gD to a cellular receptor, two, MC2 and MC5, block a separate, downstream step in the fusion pathway which is presumed to be the activation of the modulator of fusion, gH/gL. By combining epitope mapping of a panel of gD mutants with fusion and virus entry assays, we have identified residues that are critical in the binding and function of these two MAbs. This new information helps to define the site of the presumptive interaction of gD with gH/gL, of which we have limited knowledge.

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The Domains of Glycoprotein D Required To Block Apoptosis Induced by Herpes Simplex Virus 1 Are Largely Distinct from Those Involved in Cell-Cell Fusion and Binding to Nectin1

Journal of Virology ◽

10.1128/jvi.77.6.3759-3767.2003 ◽

2003 ◽

Vol 77 (6) ◽

pp. 3759-3767 ◽

Cited By ~ 33

Author(s):

Guoying Zhou ◽

Elisa Avitabile ◽

Gabriella Campadelli-Fiume ◽

Bernard Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Cell Fusion ◽

Ectopic Expression ◽

Full Length ◽

Glycoprotein D ◽

Herpes Simplex Virus 1 ◽

Mannose 6 Phosphate ◽

Simplex Virus ◽

Cell Cell

ABSTRACT Glycoprotein D (gD) interacts with two alternative protein receptors, nectin1 and HveA, to mediate herpes simplex virus (HSV) entry into cells. Fusion of the envelope with the plasma membrane requires, in addition to gD, glycoproteins gB, gH, and gL. Coexpression of the four glycoproteins (gD, gB, gH, and gL) promotes cell-cell fusion. gD delivered in trans is also capable of blocking the apoptosis induced by gD deletion viruses grown either in noncomplementing cells (gD−/−) or in complementing cells (gD−/+). While ectopic expression of cation-independent mannose-6 phosphate receptor blocks apoptosis induced by both stocks, other requirements differ. Thus, apoptosis induced by gD−/− virus is blocked by full-length gD (or two gD fragments reconstituting a full-length molecule), whereas ectopic expression of the gD ectodomain is sufficient to block apoptosis induced by gD−/+ virus. In this report we took advantage of a set of gD insertion-deletion mutants to map the domains of gD required to block apoptosis by gD−/− and gD−/+ viruses and those involved in cell-cell fusion. The mutations that resulted in failure to block apoptosis were the same for gD−/− and gD−/+ viruses and were located in three sites, one within the immunoglobulin-type core region (residues 125, 126, and 151), one in the upstream connector region (residues 34 and 43), and one in the C-terminal portion of the ectodomain (residue 277). A mutant that carried amino acid substitutions at the three glycosylation sites failed to block apoptosis but behaved like wild-type gD in all other assays. The mutations that inhibited polykaryocyte formation were located in the upstream connector region (residues 34 and 43), at the α1 helix (residue 77), in the immunoglobulin core and downstream regions (residue 151 and 187), and at the α3 helix (residues 243 and 246). Binding of soluble nectin1-Fc to cells expressing the mutant gDs was generally affected by the same mutations that affected fusion, with one notable exception (Δ277-310), which affected fusion without hampering nectin1 binding. This deletion likely identifies a region of gD involved in fusion activity at a post-nectin1-binding step. We conclude that whereas mutations that affected all functions (e.g., upstream connector region and residue 151) may be detrimental to overall gD structure, the mutations that affect specific activities identify domains of gD involved in the interactions with entry receptors and fusogenic glycoproteins and with cellular proteins required to block apoptosis. The evidence that glycosylation of gD is required for blocking apoptosis supports the conclusion that the interacting protein is the mannose-6 phosphate receptor.

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Herpes B Virus Utilizes Human Nectin-1 but Not HVEM or PILRα for Cell-Cell Fusion and Virus Entry

Journal of Virology ◽

10.1128/jvi.00041-12 ◽

2012 ◽

Vol 86 (8) ◽

pp. 4468-4476 ◽

Cited By ~ 20

Author(s):

Qing Fan ◽

Melanie Amen ◽

Mallory Harden ◽

Alberto Severini ◽

Anthony Griffiths ◽

...

Keyword(s):

Cell Fusion ◽

Virus Entry ◽

B Virus ◽

Herpes B Virus ◽

Cell Cell

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Murine Coronavirus Requires Lipid Rafts for Virus Entry and Cell-Cell Fusion but Not for Virus Release

Journal of Virology ◽

10.1128/jvi.79.15.9862-9871.2005 ◽

2005 ◽

Vol 79 (15) ◽

pp. 9862-9871 ◽

Cited By ~ 55

Author(s):

Keum S. Choi ◽

Hideki Aizaki ◽

Michael M. C. Lai

Keyword(s):

Plasma Membrane ◽

Lipid Rafts ◽

Cell Fusion ◽

Lipid Raft ◽

Virus Entry ◽

Buoyant Density ◽

Spike Protein ◽

Cholesterol Depletion ◽

Virus Binding ◽

Cell Cell

ABSTRACT Thorp and Gallagher first reported that depletion of cholesterol inhibited virus entry and cell-cell fusion of mouse hepatitis virus (MHV), suggesting the importance of lipid rafts in MHV replication (E. B. Thorp and T. M. Gallagher, J. Virol. 78:2682-2692, 2004). However, the MHV receptor is not present in lipid rafts, and anchoring of the MHV receptor to lipid rafts did not enhance MHV infection; thus, the mechanism of lipid rafts involvement is not clear. In this study, we defined the mechanism and extent of lipid raft involvement in MHV replication. We showed that cholesterol depletion by methyl β-cyclodextrin or filipin did not affect virus binding but reduced virus entry. Furthermore, MHV spike protein bound to nonraftraft membrane at 4°C but shifted to lipid rafts at 37°C, indicating a redistribution of membrane following virus binding. Thus, the lipid raft involvement in MHV entry occurs at a step following virus binding. We also found that the viral spike protein in the plasma membrane of the infected cells was associated with lipid rafts, whereas that in the Golgi membrane, where MHV matures, was not. Moreover, the buoyant density of the virion was not changed when MHV was produced from the cholesterol-depleted cells, suggesting that MHV does not incorporate lipid rafts into the virion. These results indicate that MHV release does not involve lipid rafts. However, MHV spike protein has an inherent ability to associate with lipid rafts. Correspondingly, cell-cell fusion induced by MHV was retarded by cholesterol depletion, consistent with the association of the spike protein with lipid rafts in the plasma membrane. These findings suggest that MHV entry requires specific interactions between the spike protein and lipid rafts, probably during the virus internalization step.

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Blockade of SARS-CoV-2 spike protein-mediated cell–cell fusion using COVID-19 convalescent plasma

Scientific Reports ◽

10.1038/s41598-021-84840-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ling Wang ◽

Juan Zhao ◽

Lam N. T. Nguyen ◽

James L. Adkins ◽

Madison Schank ◽

...

Keyword(s):

Membrane Fusion ◽

Cell Fusion ◽

Neutralizing Antibodies ◽

Molecular Mechanisms ◽

Virus Entry ◽

High Capacity ◽

Target Cells ◽

Global Public Health ◽

S Protein ◽

Cell Cell

AbstractThe recent COVID-19 pandemic poses a serious threat to global public health, thus there is an urgent need to define the molecular mechanisms involved in SARS-CoV-2 spike (S) protein-mediated virus entry that is essential for preventing and/or treating this emerging infectious disease. In this study, we examined the blocking activity of human COVID-19 convalescent plasma by cell–cell fusion assays using SARS-CoV-2-S-transfected 293 T as effector cells and ACE2-expressing 293 T as target cells. We demonstrate that the SARS-CoV-2 S protein exhibits a very high capacity for membrane fusion and is efficient in mediating virus fusion and entry into target cells. Importantly, we find that COVID-19 convalescent plasma with high titers of IgG neutralizing antibodies can block cell–cell fusion and virus entry by interfering with the SARS-CoV-2-S/ACE2 or SARS-CoV-S/ACE2 interactions. These findings suggest that COVID-19 convalescent plasma may not only inhibit SARS-CoV-2-S but also cross-neutralize SARS-CoV-S-mediated membrane fusion and virus entry, supporting its potential as a preventive and/or therapeutic agent against SARS-CoV-2 as well as other SARS-CoV infections.

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The Neutralizing Linear Epitope of Human Herpesvirus 6A Glycoprotein B Does Not Affect Virus Infectivity

Journal of Virology ◽

10.1128/jvi.02074-17 ◽

2017 ◽

Vol 92 (5) ◽

Cited By ~ 2

Author(s):

Aika Wakata ◽

Satoshi Kanemoto ◽

Huamin Tang ◽

Akiko Kawabata ◽

Mitsuhiro Nishimura ◽

...

Keyword(s):

Amino Acids ◽

Monoclonal Antibody ◽

Cell Fusion ◽

Virus Entry ◽

Human Herpesvirus ◽

Homology Model ◽

Glycoprotein B ◽

Linear Epitope ◽

Epitope Residue ◽

Cell Cell

ABSTRACTHuman herpesvirus 6A (HHV-6A) glycoprotein B (gB) is a glycoprotein consisting of 830 amino acids and is essential for the growth of the virus. Previously, we reported that a neutralizing monoclonal antibody (MAb) called 87-y-13 specifically reacts with HHV-6A gB, and we identified its epitope residue at asparagine (Asn) 347 on gB. In this study, we examined whether the epitope recognized by the neutralizing MAb is essential for HHV-6A infection. We constructed HHV-6A bacterial artificial chromosome (BAC) genomes harboring substitutions at Asn347, namely, HHV-6A BACgB(N347K) and HHV-6A BACgB(N347A). These mutant viruses could be reconstituted and propagated in the same manner as the wild type and their revertants, and MAb 87-y-13 could not inhibit infection by either mutant. In a cell-cell fusion assay, Asn at position 347 on gB was found to be nonessential for cell-cell fusion. In addition, in building an HHV-6A gB homology model, we found that the epitope of the neutralizing MAb is located on domain II of gB and is accessible to solvents. These results indicate that Asn at position 347, the linear epitope of the neutralizing MAb, does not affect HHV-6A infectivity.IMPORTANCEGlycoprotein B (gB) is one of the most conserved glycoproteins among all herpesviruses and is a key factor for virus entry. Therefore, antibodies targeted to gB may neutralize virus entry. Human herpesvirus 6A (HHV-6A) encodes gB, which is translated to a protein of about 830 amino acids (aa). Using a monoclonal antibody (MAb) for HHV-6A gB, which has a neutralizing linear epitope, we analyzed the role of its epitope residue, N347, in HHV-6A infectivity. Interestingly, this gB linear epitope residue, N347, was not essential for HHV-6A growth. By constructing a homology model of HHV-6A gB, we found that N347 was located in the region corresponding to domain II. Therefore, with regard to its neutralizing activity against HHV-6A infection, the epitope on gB might be exposed to solvents, suggesting that it might be a target of the immune system.

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Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

Journal of Virology ◽

10.1128/jvi.01805-07 ◽

2008 ◽

Vol 82 (6) ◽

pp. 2883-2894 ◽

Cited By ~ 25

Author(s):

Megan W. Howard ◽

Emily A. Travanty ◽

Scott A. Jeffers ◽

M. K. Smith ◽

Sonia T. Wennier ◽

...

Keyword(s):

Amino Acids ◽

Cell Fusion ◽

Transmembrane Domain ◽

Virus Entry ◽

Aromatic Amino Acids ◽

Wild Type ◽

Aromatic Residues ◽

Wild Type Level ◽

S Proteins ◽

Cell Cell

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein (S) is a class I viral fusion protein that binds to its receptor glycoprotein, human angiotensin converting enzyme 2 (hACE2), and mediates virus entry and cell-cell fusion. The juxtamembrane domain (JMD) of S is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses. Alanine substitutions for one or two of the six aromatic residues in the JMD did not alter the surface expression of the SARS-CoV S proteins with a deletion of the C-terminal 19 amino acids (S Δ19) or reduce binding to soluble human ACE2 (hACE2). However, hACE2-dependent entry of trypsin-treated retrovirus pseudotyped viruses expressing JMD mutant S Δ19 proteins was greatly reduced. Single alanine substitutions for aromatic residues reduced entry to 10 to 60% of the wild-type level. The greatest reduction was caused by residues nearest the transmembrane domain. Four double alanine substitutions reduced entry to 5 to 10% of the wild-type level. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60 to 70% of the wild-type level for all single alanine substitutions and the Y1188A/Y1191A protein. S Δ19 proteins with other double alanine substitutions reduced cell-cell fusion further, from 40% to less than 20% of wild-type levels. The aromatic amino acids in the JMD of the SARS-CoV S glycoprotein play critical roles in receptor-dependent virus-cell and cell-cell fusion. Because the JMD is so highly conserved in all coronavirus S proteins, it is a potential target for development of drugs that may inhibit virus entry and/or cell-cell fusion mediated by S proteins of all coronaviruses.

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